In testing for analytes such as drug molecules, immunoassays have proven to be especially useful. In an immunoassay, the interaction of an analyte, sometimes referred to as an antigen, with a specific receptor, typically an antibody, results in the formation of an antigen-antibody complex. This complex can be detected by various measurements, such as radioactivity, fluorescence, light absorption and light scattering. The results are then correlated with the presence, absence, and ideally the concentration of the analyte.
One type of immunoassay is the particle-based agglutination immunoassay, which is based on the binding of an antigen with an antibody, one of which is bound to a particle. The particles employed are often polymer particles, such as polystyrene and poly(methyl methacrylate), which are typically produced by an emulsion polymerization process. Other particle systems may also be used, including gold particles such as gold nanoparticles and gold colloids; and ceramic particles, such as silica, glass, and metal oxide particles. The binding agent, which is an antigen or an antibody, may be physically adsorbed onto the particle; however, greater stability and longer shelf-life are obtained when the binding agent is covalently attached. See for example J. L. Ortega-Vinuesa et al. J. Biomater. Sci. Polymer Edn., 12(4), 379-408 (2001).
Particles having covalently linked binding agents are typically prepared by activation of the particles, followed by coupling of the binding agent to the activated particles. In some instances, the activation is followed by a coupling of a linking group to the activated particles, and the linking group can then be used to tether the binding agent to the particle. Linking groups include, for example, avidin or streptavidin and chemical moieties presenting functional groups such as maleimides and thiols.
For particles having carboxylate groups bound to the surface, activation is often achieved by contacting the particles with a solution of a carbodiimide coupling reagent and a succinimide reagent such as N-hydroxysuccinimide (NHS) or N-hydroxysulfosuccinimide (sNHS). The carboxylate groups on the surface are thus converted into NHS-ester or sNHS-ester groups. Carbodiimide couplers include, for example, N-ethyl-Nxe2x80x2-(3-dimethyl-aminopropyl)carbodiimide (EDC); dicyclohexylcarbodiimide (DCC); and diisopropylcarbodiimide (DIC). A binding agent or a linking group can then be coupled to the particles by mixing the activated particles and the binding agent or linking group in an aqueous mixture. Sensitized particles are formed once a binding agent has been linked to the surface, whether by contact with activated particles or by reaction with particles containing linking groups. An illustration of a simple version of this process, using NHS as the succinimide reagent and sensitizing with an antibody, is given in the following reaction scheme. The sensitized particles produced by this process are then typically treated with a blocking agent, for example bovine serum albumin (BSA), which serves to react with and quench remaining NHS or sNHS ester groups. 
When such sensitized particles are mixed in an aqueous environment with a sample to be analyzed, the analyte in the sample will specifically bind to the antibody, which may be present on the particle or as a separate entity in the liquid mixture. This interaction may cause the particles to agglutinate (direct recognition), or it may hinder an agglutination process (competitive inhibition), depending on the particular format of the assay. Agglutination is the formation of clusters of particles having a larger collective size than that of an individual particle, and can be detected by measuring the change in the absorbance or the scattering of light by the sample. Ideally, the degree of agglutination in a particle-based agglutination immunoassay can be correlated with the amount of antigen in the sample. However, non-specific interactions between the particles and the sample can result in agglutination or inhibition of agglutination of the particles which is unrelated to the antigen-antibody interaction. These unwanted interactions can cause false positive or false negative results, and can also lead to inaccurate correlations of the agglutination response to the concentration of the antigen of interest. All of these undesirable effects compromise the quality of the assay result, and are collectively known as xe2x80x9cinterference.xe2x80x9d
Various substances have been reported to reduce interference in agglutination immunoassays by suppressing non-specific interactions. For example, U.S. Pat. No. 4,362,531 describes the use of salts such as guanidinium salts, thiocyanate salts, and alkali metal halide salts. In addition, U.S. Pat. Nos. 5,506,151 and 5,486,479 describe the use of primary, secondary, and tertiary amines such as 1-ethyl-3-(3-dimethylaminopropyl)urea (EDU), 3-dimethylaminopropylamine, 3-diethylaminopropylamine, dimethylaminopropylchloride, and 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMC). Other substances reported include halogen substituted carboxylic acids (U.S. Pat. No. 4,536,478) and substituted amides (U.S. Pat. No. 4,292,038). In general, these additives do not provide an optimum balance between reducing the interference due to non-specific interactions and minimizing the reactivity of the additive towards the antibody or antigen of interest. Non-specific interactions between the additive and the antibody or antigen can also contribute to interference in the immunoassay.
It is thus desirable, in particle-based agglutination immunoassays, to prevent non-specific interactions between the particles and components of the sample being analyzed. It is also desirable to provide additives which can suppress non-specific interactions within the immunoassay while avoiding interactions with the antigen or antibody.
In one aspect of the invention there is a reagent for use in immunoassays, comprising a plurality of particles and a tertiary amine compound of formula (I)
N(R1xe2x80x94X)(R2xe2x80x94Y)(R3xe2x80x94Z)xe2x80x83xe2x80x83(I); 
wherein R1, R2, and R3 are independently selected from the group consisting of alkyl and alkyl ether; and X, Y, and Z are independently selected from the group consisting of xe2x80x94OH, xe2x80x94Oxe2x80x94R4, xe2x80x94Sxe2x80x94R4, xe2x80x94C(xe2x95x90O)xe2x80x94OH, xe2x80x94C(xe2x95x90O)xe2x80x94OR4, or xe2x80x94C(xe2x95x90O)xe2x80x94NHR4, wherein R4 is alkyl. Each of said particles comprises a surface having been activated by a carbodiimide, and a binding agent linked to the surface through a covalent bond.
In another aspect of the invention, there is a reagent for use in immunoassays, comprising a plurality of particles and a tertiary amine compound of formula (II)
N(R1xe2x80x94OH)(R2xe2x80x94OH)(R3xe2x80x94OH)xe2x80x83xe2x80x83(II); 
wherein R1, R2, and R3 are independently alkyl groups comprising from 1 to 5 carbon atoms; wherein the reagent forms an assay mixture when mixed with a sample, such that the tertiary amine compound is present in the assay mixture in a concentration of 50 mM or less. Each of said particles comprises a surface having been activated by a carbodiimide, and a binding agent linked to the surface through a covalent bond.
In yet another aspect of the invention there is an assay method for determining an analyte, comprising combining a sample suspected of containing said analyte with any of the above reagents, and determining the presence or amount of said detectable complex as a measure of said analyte in said sample. The reagent comprises the antibody of said analyte, and the reagent is capable of forming a detectable complex with said analyte.
In yet another aspect of the invention there is provided a test kit, comprising any of the above reagents.
In yet another aspect of the invention there is provided, in an immunoassay method wherein a sample suspected of containing an analyte is combined with a plurality of particles, each of said particles having a surface having been activated by a carbodiimide, and a binding agent bound to the surface through a covalent bond; the improvement comprising adding to the sample, to form an assay mixture, a tertiary amine compound of formula (I)
N(R1xe2x80x94X)(R2xe2x80x94Y)(R3xe2x80x94Z)xe2x80x83xe2x80x83(I); 
wherein R1, R2, and R3 are independently selected from the group consisting of alkyl and alkyl ether; and X, Y, and Z are independently selected from the group consisting of xe2x80x94OH, xe2x80x94Oxe2x80x94R4, xe2x80x94Sxe2x80x94R4, xe2x80x94C(xe2x95x90O)xe2x80x94OH, xe2x80x94C(xe2x95x90O)xe2x80x94OR4, or xe2x80x94C(xe2x95x90O)xe2x80x94NHR4, wherein R4 is alkyl.